Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis

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Kidney fibrosis is marked by an epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs). Here we find that, during renal fibrosis, TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of the EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several solute and solvent transporters in TECs. We also found that transgenic expression of either Twist1 (encoding twist family bHLH transcription factor 1, known as Twist) or Snai1 (encoding snail family zinc finger 1, known as Snail) expression is sufficient to promote prolonged TGF-β1–induced G2 arrest of TECs, limiting the cells' potential for repair and regeneration. In mouse models of experimentally induced renal fibrosis, conditional deletion of Twist1 or Snai1 in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while also restoring cell proliferation, dedifferentiation-associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of the EMT program in TECs during chronic renal injury represents a potential anti-fibrosis therapy.

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Figure 1: Genetic targeting of EMT reduces renal fibrosis and improves TEC health.
Figure 2: Inhibition of the EMT prevents loss of TEC-associated solute and solvent transporters.
Figure 3: An EMT program is associated with deregulated expression and functionality of TEC solute and solvent transporters in humans with kidney disease.
Figure 4: Inhibition of an EMT reduces immune infiltration in kidney fibrosis.
Figure 5: EMT program G2 cell cycle arrest.
Figure 6: p21 controls the EMT program G2 cell cycle arrest.

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This work was primarily supported with funds from the University of Texas M.D. Anderson Cancer Center (UT MDACC), partially supported by the Cancer Prevention and Research Institute of Texas and funding from the University of Texas System Science and Technology Acquisition and Retention (STARS) awards to RK and VSL. The research in R.K. laboratory is also supported by the US National Institutes of Health (NIH) (grants CA-155370, CA-151925, DK-081576, DK-55001) and the Metastasis Research Center at the M.D. Anderson Cancer Center (P30CA016672). V.S.L. is supported by the NIH under award number P30CA016672 and the Khalifa Bin Zayed Al Nahya Foundation. This research was performed in the Flow Cytometry & Cellular Imaging Facility at UT MDACC, which is supported in part by the NIH through MDACC Support grant CA–016672. This work was in part supported by the Deutsche Forschungsgemeinschaft (equipment grant INST1525/16–1 FUGG). SnailloxP/loxP mice were kindly provided by S.J. Weiss, University of Michigan, and Twist1loxP/loxP mice were kindly provided by R.R. Behringer, UT MDACC via the Mutant Mouse Regional Resource Center (MMRRC) repository. pcDNA3-Twist plasmid was kindly provided by R. Maestro, Centro di Riferimento Oncologico National Cancer Institute, Italy. Nephrotoxic serum was a kind gift from D.J. Salant, Boston University. MCT cells were a gift from E.G. Neilson, Northwestern University School of Medicine. We thank E. Lawson for technical help with immunostaining, E. Chang for help with digital microscopy scanning of tissue histology slides and L. Gibson for help with breeding and genotyping mice.

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R.K. conceptually designed the strategy for this study, participated in discussions, provided intellectual input, supervised the studies and wrote the manuscript. V.S.L. designed the study, provided intellectual input, supervised and conducted the studies, designed and performed experiments, generated the figures and wrote the manuscript. S.L. designed and performed experiments, collected the data, generated the figures and participated in writing the manuscript. S.L., V.S.L., B.T., H.S., K.V., J.L.C., C.-C.W.,Y.H., B.C.B., T.P.-H., and H.N. performed some experiments and collected data. The data was analyzed by S.L., V.S.L., B.T., J.L.C., C.-C.W. and T.P.-H. J.P.A. and M.Z. participated in discussions, provided intellectual input, supervised the studies and edited the manuscript.

Correspondence to Raghu Kalluri.

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J.P.A. is an inventor of intellectual property owned by the University of California, Berkeley, and licensed to Bristol Meyers–Squibb.

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Lovisa, S., LeBleu, V., Tampe, B. et al. Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med 21, 998–1009 (2015) doi:10.1038/nm.3902

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